Little is known about the molecular details of RNA replication for many RNA viruses, yet it is of primary interest for understanding basic molecular principles and the molecular biology of these viruses. Information gained through this understanding can help identify major targets for antiviral drugs and provide insight toward development of RNA viruses as safe vectors. The studies proposed herein will examine aspects of coronavirus RNA replication. Coronaviruses are widespread, medically important respiratory and enteric pathogens of humans and a broad range of domestic animals. The viruses are positive(+)-sense, single-stranded, enveloped RNA viruses whose genome is the largest (-30 kb) of all the RNA viruses. Coronaviruses express their genetic information in a unique manner, and the mechanisms and regulation of RNA replication are poorly understood but highly desired. Mouse hepatitis coronavirus (MIHV) defective interfering (DI) RNAs, targeted RNA recombination with the MBV genome and a new transmissible gastroenteritis coronavirus (TGEV) infectious cDNA clone will be used in our studies. The specific aims of this proposal are: (1) To determine the promoter sequence and structural requirements for coronavirus RNA negative-strand synthesis. (2) To identify the other host proteins (beyond (PABP) that specifically bind the 3' UTR. (3) To study the role of translation in coronavirus RNA replication. These studies are based on the premise that protein interactions with specific elements in the viral 3' UTR and poly(A) tail are important for RNA synthesis. We hypothesize that viral RNA replication is facilitated by interaction between the 3'- and 5'-ends of the genome that is established during translation. The long-term goal of our research program is to understand coronavirus RNA replication at the molecular level. The proposed studies will take us toward that goal by investigating the early steps in viral RNA replication that immediately follow (and may be set up by) RNA translation in the host cells. In addition our studies should contribute to understanding of protein-RNA interactions and the coupling of fundamental cellular processes, like translation.